Amit P. Bhavsar

Exome Sequencing and the Management of Neurometabolic Disorders

BACKGROUND Whole-exome sequencing has transformed gene discovery and diagnosis in rare diseases. Translation into disease-modifying treatments is challenging, particularly for intellectual developmental disorder. However, the exception is inborn errors of metabolism, since many of these disorders are responsive to therapy that targets pathophysiological features at the molecular or cellular level. METHODS To uncover the genetic basis of potentially treatable inborn errors of metabolism, we combined deep clinical phenotyping (the comprehensive characterization of the discrete components of a patients clinical and biochemical phenotype) with whole-exome sequencing analysis through a semiautomated bioinformatics pipeline in consecutively enrolled patients with intellectual developmental disorder and unexplained metabolic phenotypes. RESULTS We performed whole-exome sequencing on samples obtained from 47 probands. Of these patients, 6 were excluded, including 1 who withdrew from the study. The remaining 41 probands had been born to predominantly nonconsanguineous parents of European descent. In 37 probands, we identified variants in 2 genes newly implicated in disease, 9 candidate genes, 22 known genes with newly identified phenotypes, and 9 genes with expected phenotypes; in most of the genes, the variants were classified as either pathogenic or probably pathogenic. Complex phenotypes of patients in five families were explained by coexisting monogenic conditions. We obtained a diagnosis in 28 of 41 probands (68%) who were evaluated. A test of a targeted intervention was performed in 18 patients (44%). CONCLUSIONS Deep phenotyping and whole-exome sequencing in 41 probands with intellectual developmental disorder and unexplained metabolic abnormalities led to a diagnosis in 68%, the identification of 11 candidate genes newly implicated in neurometabolic disease, and a change in treatment beyond genetic counseling in 44%. (Funded by BC Childrens Hospital Foundation and others.).

A coding variant in RARG confers susceptibility to anthracycline-induced cardiotoxicity in childhood cancer.

Anthracyclines are used in over 50% of childhood cancer treatment protocols, but their clinical usefulness is limited by anthracycline-induced cardiotoxicity (ACT) manifesting as asymptomatic cardiac dysfunction and congestive heart failure in up to 57% and 16% of patients, respectively. Candidate gene studies have reported genetic associations with ACT, but these studies have in general lacked robust patient numbers, independent replication or functional validation. Thus, the individual variability in ACT susceptibility remains largely unexplained. We performed a genome-wide association study in 280 patients of European ancestry treated for childhood cancer, with independent replication in similarly treated cohorts of 96 European and 80 non-European patients. We identified a nonsynonymous variant (rs2229774, p.Ser427Leu) in RARG highly associated with ACT (P = 5.9 × 10−8, odds ratio (95% confidence interval) = 4.7 (2.7–8.3)). This variant alters RARG function, leading to derepression of the key ACT genetic determinant Top2b, and provides new insight into the pathophysiology of this severe adverse drug reaction.

The Salmonella Type III Effector SspH2 Specifically Exploits the NLR Co-chaperone Activity of SGT1 to Subvert Immunity

To further its pathogenesis, S. Typhimurium delivers effector proteins into host cells, including the novel E3 ubiquitin ligase (NEL) effector SspH2. Using model systems in a cross-kingdom approach we gained further insight into the molecular function of this effector. Here, we show that SspH2 modulates innate immunity in both mammalian and plant cells. In mammalian cell culture, SspH2 significantly enhanced Nod1-mediated IL-8 secretion when transiently expressed or bacterially delivered. In addition, SspH2 also enhanced an Rx-dependent hypersensitive response in planta. In both of these nucleotide-binding leucine rich repeat receptor (NLR) model systems, SspH2-mediated phenotypes required its catalytic E3 ubiquitin ligase activity and interaction with the conserved host protein SGT1. SGT1 has an essential cell cycle function and an additional function as an NLR co-chaperone in animal and plant cells. Interaction between SspH2 and SGT1 was restricted to SGT1 proteins that have NLR co-chaperone function and accordingly, SspH2 did not affect SGT1 cell cycle functions. Mechanistic studies revealed that SspH2 interacted with, and ubiquitinated Nod1 and could induce Nod1 activity in an agonist-independent manner if catalytically active. Interestingly, SspH2 in vitro ubiquitination activity and protein stability were enhanced by SGT1. Overall, this work adds to our understanding of the sophisticated mechanisms used by bacterial effectors to co-opt host pathways by demonstrating that SspH2 can subvert immune responses by selectively exploiting the functions of a conserved host co-chaperone.

Recommendations for genetic testing to reduce the incidence of anthracycline‐induced cardiotoxicity

Anthracycline‐induced cardiotoxicity (ACT) occurs in 57% of treated patients and remains an important limitation of anthracycline‐based chemotherapy. In various genetic association studies, potential genetic risk markers for ACT have been identified. Therefore, we developed evidence‐based clinical practice recommendations for pharmacogenomic testing to further individualize therapy based on ACT risk.

Mitochondrial Carbonic Anhydrase VA Deficiency Resulting from CA5A Alterations Presents with Hyperammonemia in Early Childhood

Four children in three unrelated families (one consanguineous) presented with lethargy, hyperlactatemia, and hyperammonemia of unexplained origin during the neonatal period and early childhood. We identified and validated three different CA5A alterations, including a homozygous missense mutation (c.697T>C) in two siblings, a homozygous splice site mutation (c.555G>A) leading to skipping of exon 4, and a homozygous 4 kb deletion of exon 6. The deleterious nature of the homozygous mutation c.697T>C (p.Ser233Pro) was demonstrated by reduced enzymatic activity and increased temperature sensitivity. Carbonic anhydrase VA (CA-VA) was absent in liver in the child with the homozygous exon 6 deletion. The metabolite profiles in the affected individuals fit CA-VA deficiency, showing evidence of impaired provision of bicarbonate to the four enzymes that participate in key pathways in intermediary metabolism: carbamoylphosphate synthetase 1 (urea cycle), pyruvate carboxylase (anaplerosis, gluconeogenesis), propionyl-CoA carboxylase, and 3-methylcrotonyl-CoA carboxylase (branched chain amino acids catabolism). In the three children who were administered carglumic acid, hyperammonemia resolved. CA-VA deficiency should therefore be added to urea cycle defects, organic acidurias, and pyruvate carboxylase deficiency as a treatable condition in the differential diagnosis of hyperammonemia in the neonate and young child.

Quantitative Mass Spectrometry Catalogues Salmonella Pathogenicity Island-2 Effectors and Identifies Their Cognate Host Binding Partners

Gram-negative bacterial pathogens have developed specialized secretion systems to transfer bacterial proteins directly into host cells. These bacterial effectors are central to virulence and reprogram host cell processes to favor bacterial survival, colonization, and proliferation. Knowing the complete set of effectors encoded by a particular pathogen is the key to understanding bacterial disease. In addition, the identification of the molecular assemblies that these effectors engage once inside the host cell is critical to determining the mechanism of action of each effector. In this work we used stable isotope labeling of amino acids in cell culture (SILAC), a powerful quantitative proteomics technique, to identify the proteins secreted by the Salmonella pathogenicity island-2 type three secretion system (SPI-2 T3SS) and to characterize the host interaction partners of SPI-2 effectors. We confirmed many of the known SPI-2 effectors and were able to identify several novel substrate candidates of this secretion system. We verified previously published host protein-effector binding pairs and obtained 11 novel interactions, three of which were investigated further and confirmed by reciprocal co-immunoprecipitation. The host cell interaction partners identified here suggest that Salmonella SPI-2 effectors target, in a concerted fashion, cellular processes such as cell attachment and cell cycle control that are underappreciated in the context of infection. The technology outlined in this study is specific and sensitive and serves as a robust tool for the identification of effectors and their host targets that is readily amenable to the study of other bacterial pathogens.

The emerging era of pharmacogenomics: current successes, future potential, and challenges

The vast range of genetic diversity contributes to a wonderful array of human traits and characteristics. Unfortunately, a consequence of this genetic diversity is large variability in drug response between people, meaning that no single medication is safe and effective in everyone. The debilitating and sometimes deadly consequences of adverse drug reactions (ADRs) are a major and unmet problem of modern medicine. Pharmacogenomics can uncover associations between genetic variation and drug safety and has the potential to predict ADRs in individual patients. Here we review pharmacogenomic successes leading to changes in clinical practice, as well as clinical areas probably to be impacted by pharmacogenomics in the near future. We also discuss some of the challenges, and potential solutions, that remain for the implementation of pharmacogenomic testing into clinical practice for the significant improvement of drug safety.

Proteomics as a probe of microbial pathogenesis and its molecular boundaries

Proteomic technology offers an unprecedented systematic approach to investigate the protein complement of any organism. The field of microbial pathogenesis has greatly benefited from other systems approaches, and the application of proteomics to the study of infectious agents is beginning to emerge. Such applications include unambiguously identifying complete virulence factor inventories, studying the response of both host and pathogen to the infection process and elucidating mechanistic actions of virulence factors as they interface with host cells. This review will highlight examples where proteomic studies have contributed to our understanding of pathogenesis in these areas, with an emphasis on pathogens that employ type III and type IV secretion systems. In addition, we will discuss areas where proteomics may help shape further investigation and discovery in this field.

Global Impact of Salmonella Pathogenicity Island 2-secreted Effectors on the Host Phosphoproteome

During the late stages of infection, Salmonella secretes numerous effectors through a type III secretion system that is encoded within Salmonella pathogenicity island 2 (SPI2). Despite the importance of SPI2 as a major virulence factor leading to the systemic spread of the bacteria and diseases, a global view of its effects on host responses is still lacking. Here, we measured global impacts of SPI2 effectors on the host phosphorylation and protein expression levels in RAW264.7 and in HeLa cells, as macrophage and nonphagocytic models of infection. We observe that SPI2 effectors differentially modulate the host phosphoproteome and cellular processes (e.g. protein trafficking, cytoskeletal regulation, and immune signaling) in a host cell-dependent manner. Our unbiased approach reveals the involvement of many previously unrecognized proteins, including E3 ligases (HERC4, RanBP2, and RAD18), kinases (CDK, SIK3, and WNK1), and histones (H2B1F, H4, and H15), in late stages of Salmonella infection. Furthermore, from this phosphoproteome analysis and other quantitative screens, we identified HSP27 as a direct in vitro and in vivo molecular target of the only type III secreted kinase, SteC. Using biochemical and cell biological assays, we demonstrate that SteC phosphorylates multiple sites in HSP27 and induces actin rearrangement through this protein. Together, these results provide a broader landscape of host players contributing to specific processes/pathways mediated by SPI2 effectors than was previously appreciated.

Two Conserved Histidine Residues Are Critical to the Function of the TagF-like Family of Enzymes

The TagF protein from Bacillus subtilis 168 is the poly(glycerol phosphate) polymerase responsible for the synthesis of wall teichoic acid and is the prototype member of a poorly understood family of similar teichoic acid synthetic enzymes. Here we describe in vitro and in vivo characterization of TagF, which localizes the active site to the carboxyl terminus of the protein and identifies residues that are critical for catalysis. We also establish the first mechanistic link among TagF and similar proteins by demonstrating that the identified residues are also critical in the function of TagB, a homologous enzyme implicated as the glycerophosphotransferase responsible for priming poly(glycerol phosphate) synthesis (Bhavsar, A. P., Truant, R., and Brown, E. D. (Sept. 2, 2005) J. Biol. Chem. 36691–36700). We investigated the dependence of TagF activity on pH and showed that deprotonation of a residue with a pKa near neutral is critical for proper function. Alteration of histidine residues 474 and 612 by site-directed mutagenesis abolished TagF activity in vitro (5000-fold reduction in kcat/Km) while variants in four other conserved acidic residues showed minimal loss of activity. Complementation using H474A and H612A mutant alleles failed to suppress a lethal temperature-sensitive tagF defect in vivo despite confirmation of robust expression by Western blot. When corresponding mutations were made to the homologous tagB gene, these alleles were unable to suppress a tagB temperature-sensitive lethal phenotype. These results extend the mechanistic observations for TagF across a wider family of enzymes and provide the first biochemical evidence for the relatedness of these two enzymes.